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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation, but was considered not to affect the purpose or integrity of the study.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Preparation test item:
Nominal amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for 5 minutes. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates). pH was measured and adjusted to between pH 7.0 and 8.0.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage. The sample was maintained in the lab on continuous aeration at 21ºC and used at the day of collection.
The pH was 7.7 and the suspended solids concentration was 3.0 g/L.
Test type:
static
Water media type:
other: deionized reverse osmosis water
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
Exposure duration was reduced from 3 hours to 30 minutes due to the volatile nature of the test item.
Test temperature:
between 19 and 20 ºC
pH:
T0 control: 7.6-7.8
T30 min control: 7.5-7.7

T0 test item: 7.7-7.8
T30 min test item: 7.6-7.7

T0 3,5 DCP: 7.7-7.8
T30 min 3,5 DCP: 7.8
Dissolved oxygen:
After 15 min:
control: 5.4-7.0 mg/L
test item: 5.4-6.1 mg/L
reference substance: 5.6-7.1 mg/L
Nominal and measured concentrations:
Test substance
Nominal: 10, 100 and 1000 mg/L

Reference substance
Nominal: 3.2, 10 en 32 mg/L
Details on test conditions:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Finally two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 and 20 ºC.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
< 1 000 mg/L
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
< 1 000 mg/L
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
ca. 1 000 mg/L
Details on results:
The dissolved oxygen concentrations after 15 minutes contact time in all vessels were above 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L.

Statistically significant toxic effects were shown at the test concentration of 1000 mg/L. It was not possible to perform statistical analysis for the test concentrations of 10 and 100 mg/L as the oxygen consumption data for the test concentrations of 10 and 100 mg/L could not be calculated due to the initial dissolved oxygen concentrations where the rate of respiration could be measured over the linear portion of the trace falling below 2 mg O2/L. This was considered to be possibly due to the shorter study length and/or the lower rate of aeration employed in the test (0.5 L/minute) which was as a result of the volatile nature of the test item. As a result of this and the limitations of the test item it was considered appropriate not to conduct a definitive test to obtain either a NOEC or a more accurate EC50 value for the test item.
Results with reference substance (positive control):
3,5-dichlorophenol EC10 (30 minutes): 2.2 mg/I
3,5-dichlorophenol EC20 (30 minutes): 3.1 mg/I
3,5-dichlorophenol EC50 (30 minutes): 8.8 mg/I
Reported statistics and error estimates:
95% confidence limits for 3,5 DCP were for the EC50 7.0-11 mg/L

Oxygen Consumption Rates and Percentage Inhibition Values after 30 Minutes Contact Time in the Range-Finding Test

Nominal
Concentration
(mg/L)

Initial O2
Reading
(mg O2/L)

Measurement Period
(minutes)

Final O2Reading
(mg O2/L)

O2Consumption Rates
(mg O2/L/hour)

%
Inhibition

Control

R1

2.7

1

1.9

48.00

-

R2

3.4

2

1.9

45.00

-

R3

3.4

2

1.8

48.00

-

R4

3.8

2

2.1

51.00

-

Test Item

10

*

*

*

*

*

100

*

*

*

*

*

1000 R1

2.9

3

1.7

24.00

50

1000 R2

4.1

6

2.0

21.00

56

1000 R3

3.9

5

2.0

22.80

53

3,5-dichlorophenol

3.2

5.0

5

1.8

38.40

20

10

6.3

10

2.8

21.00

56

32

7.5

10

6.4

6.60

86

R1– R4= Replicates 1 to 4

*      It was not possible to calculate the oxygen consumption data for the test concentrations of 10 and 100 mg/L due to the initial dissolved oxygen concentrations falling below 2 mg O2/L. This was considered to be possibly due to the shorter study length and/or the lower rate of aeration employed in the test (0.5 L/minute) which was as a result of the volatile nature of the test item.

Validity criteria fulfilled:
yes
Conclusions:
The effect of Tetrahydromyrcenol on the respiration of activated sewage sludge micro-organisms gave a 30-Minute EC50 value of approximately 1000 mg/L.
Executive summary:

A study was performed to assess the effect of Tetrahydromyrcenol on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)". Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 30 minutes at measured temperatures of between 19 and 20 °C with the addition of a synthetic sewage as a respiratory substrate. The rates of respiration were measured electrochemically for each culture. The study period was reduced from 3 hours to 30 minutes due to the volatile nature of the test item. The effect of the test item on the respiration of activated sewage sludge gave a 30-minute EC50 value of approximately 1000 mg/L.

Description of key information

A study was performed to assess the effect of Tetrahydromyrcenol on the respiration of activated sewage sludge using OECTG 209). Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 30 minutes at measured temperatures of between 19 and 20 °C with the addition of a synthetic sewage as a respiratory substrate. The rates of respiration were measured electrochemically for each culture. The study period was reduced from 3 hours to 30 minutes due to the volatile nature of the test item. The effect of the test item on the respiration of activated sewage sludge gave a 30-minute EC50 value of approximately 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information